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• W2144639685 abstract "In fibroblasts and hepatoma cells, interleukin-1 (IL-1) treatment results in the rapid nuclear accumulation of the transcription factor NF-κB, present largely as p65 (RelA)/p50 heterodimers. It is well established that this process is dependent in large part upon the phosphorylation and subsequent degradation of the cytosolic inhibitor IκB. We looked for other IL-1-induced modifications of NF-κB components and found that, in both cell types, IL-1 stimulation led, within minutes, to phosphorylation of both NF-κB p65 and p50. Phosphorylation of p65 was sustained for at least 30 min after addition of the cytokine and occurred principally upon serine residues. Immunoprecipitates of NF-κB complexes contained an associated protein kinase, the biochemical characteristics of which were indistinguishable from casein kinase II (CKII). Purified CKII efficiently phosphorylated p65 in vitro, apparently on the same major sites that became phosphorylated in intact IL-1-treated cells. Although IL-1 treatment caused little apparent stimulation of total cellular CKII activity, the fraction that was specifically associated with NF-κB complexes was markedly elevated by the cytokine. The association of CKII with NF-κB occurred in the cytoplasm, suggesting that this phosphorylation might be involved either in control of translocation of the activated complex or in modulation of its DNA binding properties. In fibroblasts and hepatoma cells, interleukin-1 (IL-1) treatment results in the rapid nuclear accumulation of the transcription factor NF-κB, present largely as p65 (RelA)/p50 heterodimers. It is well established that this process is dependent in large part upon the phosphorylation and subsequent degradation of the cytosolic inhibitor IκB. We looked for other IL-1-induced modifications of NF-κB components and found that, in both cell types, IL-1 stimulation led, within minutes, to phosphorylation of both NF-κB p65 and p50. Phosphorylation of p65 was sustained for at least 30 min after addition of the cytokine and occurred principally upon serine residues. Immunoprecipitates of NF-κB complexes contained an associated protein kinase, the biochemical characteristics of which were indistinguishable from casein kinase II (CKII). Purified CKII efficiently phosphorylated p65 in vitro, apparently on the same major sites that became phosphorylated in intact IL-1-treated cells. Although IL-1 treatment caused little apparent stimulation of total cellular CKII activity, the fraction that was specifically associated with NF-κB complexes was markedly elevated by the cytokine. The association of CKII with NF-κB occurred in the cytoplasm, suggesting that this phosphorylation might be involved either in control of translocation of the activated complex or in modulation of its DNA binding properties. Interleukin-1 (IL-1) 1The abbreviations used are: IL-1, interleukin-1; EMSA, electrophoretic mobility shift assay; HGF, human gingival fibroblast, HIV, human immunodeficiency virus; PMSF, phenylmethylsulfonyl fluoride; PAGE, polyacrylamide gel electrophoresis; TNF, tumor necrosis factor; CKII, casein kinase II; NF-κB, nuclear factor κB. 1The abbreviations used are: IL-1, interleukin-1; EMSA, electrophoretic mobility shift assay; HGF, human gingival fibroblast, HIV, human immunodeficiency virus; PMSF, phenylmethylsulfonyl fluoride; PAGE, polyacrylamide gel electrophoresis; TNF, tumor necrosis factor; CKII, casein kinase II; NF-κB, nuclear factor κB.receptors are widely distributed on cells of many lineages (1Dower S.K. Sims J.E. Cerretti D.P. Bird T.A. Chem. Immunol. 1992; 51: 33-64Crossref PubMed Google Scholar). Thus IL-1 can exert effects on connective tissues, for example inducing mitogenesis through secretion of PDGF; effects on lymphoid cells, such as inducing up-regulation of surface immunoglobulin and secretion of other cytokines; and effects on hepatocytes, for example inducing secretion of acute phase proteins such as C-reactive protein. All of these responses are triggered through a single type of receptor (IL-1R1) (2Sims J.E. Acres R.B. Grubin C.E. McMahan C.J. Wignall J.M. March C. Dower S.K. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8946-8950Crossref PubMed Scopus (237) Google Scholar), which is the archetype for a diverse family of proteins. Included in this group are the insect Toll proteins (3Gay N. Keith F. Nature. 1991; 351: 355-356Crossref PubMed Scopus (446) Google Scholar), which function in the establishment of dorsal-ventral polarity during embryogenesis, and the product of the tobacco N-gene (4Whitman S. Dinesh-Kumar S.P. Choi D. Hehl R. Corr C. Baker B. Cell. 1994; 78: 1101-1115Abstract Full Text PDF PubMed Scopus (1042) Google Scholar), which mediates resistance to the pathogen tobacco mosaic virus. Many IL-1-responsive genes are regulated through activation of Rel family transcription factors, in particular NF-κB p65/p50 heterodimers, and indeed, ligation of Toll ultimately signals the activation of Dorsal, a Rel-related protein (5Gonzalez C.S. Levine M. Science. 1994; 264: 255-258Crossref PubMed Scopus (27) Google Scholar). NF-κB is a term used to refer to a group of transcription factors/DNA binding activities that recognize a consensus motif, 5′-GGGRNNYYCC-3′. All of these factors are dimers composed of subunits that share a common domain prototypically found in c-Rel (6Siebenlist U. Franzoso G. Brown K. Annu. Rev. Cell Biol. 1994; 10: 405-455Crossref PubMed Scopus (2003) Google Scholar). The transactivating activity of the NF-κB factors is regulated by a distinct set of proteins termed IκBs, which are characterized by a series of ankyrin repeats. IκBs function by sequestering NF-κB dimers in the cytosol. (7Baldwin A.S. Annu Rev. Immunol. 1996; 14: 649-681Crossref PubMed Scopus (5515) Google Scholar). A variety of stimuli can induce activation of NF-κB (8Liou H.C. Baltimore D. Curr. Opin. Cell Biol. 1993; 5: 477-487Crossref PubMed Scopus (516) Google Scholar), including viruses, lipopolysaccharides, and pro-inflammatory cytokines such as IL-1 and tumor necrosis factor (TNF) α. Activation of NF-κB involves its translocation from the cytoplasm to the nucleus, following dissociation from one or more IκBs. Cytokines bring about this dissociation by activating a newly identified protein kinase complex (9Chen Z.J. Parent L. Maniatis T. Cell. 1996; 84: 853-862Abstract Full Text Full Text PDF PubMed Scopus (864) Google Scholar) that phosphorylates IκB at two critical residues located at the amino terminus (residues Ser-32 and Ser-36 in the case of IκBα, 10Brown K. Gerstberger S. Carlson L. Franzoso G. Siebenlist U. Science. 1995; 267: 1485-1488Crossref PubMed Scopus (1307) Google Scholar, 11Traenckner E.B.-M. Pahl H.L. Henkel T. Schmidt K.N. Wilk S. Baeuerle P.A. EMBO J. 1995; 14: 2876-2883Crossref PubMed Scopus (927) Google Scholar). The phosphorylated form of IκB, although still bound to p65/p50 heterodimers (10Brown K. Gerstberger S. Carlson L. Franzoso G. Siebenlist U. Science. 1995; 267: 1485-1488Crossref PubMed Scopus (1307) Google Scholar, 12Finco T.S. Beg A.A. Baldwin A.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11884-11889Crossref PubMed Scopus (292) Google Scholar, 13Lin Y.-C. Brown K. Siebenlist U. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 552-556Crossref PubMed Scopus (256) Google Scholar), is then subject to rapid proteolysis via the ubiquitin-proteasome pathway (14Chen Z.J. Hagler J. Palombella V. Melandri F. Scherer D. Ballard D. Maniatis T. Genes Dev. 1995; 9: 1586-1597Crossref PubMed Scopus (1159) Google Scholar). Removal of IκB exposes a nuclear localization sequence present in NF-κB, allowing the factor to translocate to the nucleus. In addition, several reports have shown that p50/p50 homodimers, which appear to bind to NF-κB sites but not transactivate, are regulated by a mechanism not involving cytoplasmic/nuclear translocation but rather are resident in the nucleus and regulated by a nuclear protein Bcl-3, which when complexed prevents association of this form of NF-κB with its recognition site (15Bours V. Franzoso G. Azarenko V. Park S. Kanno T. Brown K. Siebenlist U. Cell. 1993; 72: 729-739Abstract Full Text PDF PubMed Scopus (421) Google Scholar). Recently, it was reported that other subunits of NF-κB, including p65 and p50 are phosphoproteins (16Li C.-C. Korner M. Ferris D.K. Chen E. Dai R.-M. Longo D.L. Biochem. J. 1994; 303: 499-506Crossref PubMed Scopus (40) Google Scholar). Furthermore, activation of NF-κB in HeLa cells treated with H2O2, phorbol 12-myristate 13-acetate, or TNFα (17Naumann M. Scheidereit C. EMBO J. 1994; 13: 4597-4607Crossref PubMed Scopus (325) Google Scholar) or in endothelial cells treated with TNFα (18Ollivier V. Parry G.C.N. Cobb R.R. de Prost D. Mackman N. J. Biol. Chem. 1996; 271: 20828-20835Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar) is accompanied by an increased level of p65 phosphorylation. In this report, we show that treatment of fibroblasts or hepatoma cells with IL-1 also results in phosphorylation of p65 on serine residues, and we identify the kinase responsible as casein kinase II, raising the possibility that this is an additional level at which its activity is regulated. HepG2 hepatoma cells (ATCC HB-8065) and human diploid gingival fibroblasts (19Qwarnstrom E.E. Page R.C. Gillis S. Dower S.K. J. Biol. Chem. 1988; 263: 8261-8269Abstract Full Text PDF PubMed Google Scholar) were maintained as described previously (20Bird T.A. Sleath P.R. deRoos P.C. Dower S.K. Virca G.D. J. Biol. Chem. 1991; 266: 22661-22670Abstract Full Text PDF PubMed Google Scholar). Carrier-free [32P]orthophosphate was obtained from NEN Life Science Products while [γ-32P]ATP (3000 Ci/μmol) was from Amersham Corp. Purified polyclonal rabbit anti-peptide antibodies specific for NF-κB p65, NF-κB p50, and IκBα/MAD-3 together with corresponding immunizing peptides were obtained from Santa Cruz Biotechnology, Inc. Rabbit polyclonal antisera raised against synthetic peptides corresponding to amino acids 198–215 of the β-subunit and amino acids 376–391 of the α-subunit of human casein kinase II have been described previously (21Litchfield D.W. Lozeman F.J. Cicirelli M.F. Harrylock M. Ericsson L.H. Piening C.J. Krebs E.G. J. Biol. Chem. 1991; 266: 20380-20389Abstract Full Text PDF PubMed Google Scholar) and were the kind gift of Minoo Ahdieh, Department of Biochemistry, Immunex Corporation. Recombinant human IL-1α was expressed and purified as described (22Bird T.A. Kyriakis J.M. Tyshler L. Gayle M. Milne A. Virca G.D. J. Biol. Chem. 1994; 269: 31836-31844Abstract Full Text PDF PubMed Google Scholar). Recombinant human NF-κB p65 was a gift from Dr. Guido Franzoso (Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, Bethesda, MD). Casein kinase II, purified to greater than 95% from Pisaster ochraceus, was purchased from Upstate Biotechnology, Inc. Approximately 5 × 106 cells were washed with ice-cold PBS and resuspended in 400 μl of buffer A (10 mm HEPES, pH 7.9, 10 mm KCl, 0.1 mm EDTA, 0.1 mmEGTA, 1 mm dithiothreitol, 1 mmphenylmethlysulfonyl flouride, 1 mm leupeptin). After 15 min at 4 °C, 25 μl of 10% Nonidet P-40 was added. Cells were vortexed briefly, nuclei were pelleted by microcentrifugation, and supernatants were removed (cytoplasmic extracts). Pellets was resuspended in 200 μl of buffer B (20 mm HEPES, pH 7.9, 0.4 m NaCl, 1 mm EDTA, 1 mm EGTA, 1 mm dithiothreitol, 1 mm phenylmethlysulfonyl flouride, and 1 mm leupeptin). After 30 min at 4 °C, lysates were centrifuged and supernatant was removed (nuclear extract). Protein concentration of extracts was measured using the BCA assay (Pierce). Nuclear extracts (10 μg) were mixed with 0.02 units of poly(dI-dC) (Pharmacia Biotech Inc.) and end-labeled NF-κB oligonucleotide probe (23Sims J.E. Gayle M.A. Slack J.L. Alderson M.R. Bird T.A. Giri J.G. Colotta F.R.F. Mantovani A. Shanebeck K. Grabstein K.H. Dower S.K. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6155-6159Crossref PubMed Scopus (543) Google Scholar) (1 × 104-2.5 × 104 cpm per reaction) in binding buffer (2.5 mmHEPES, pH 7.9, 50 mm KCl, 0.1 mm EDTA, 1.8 mm dithiothreitol, 10% glycerol) and were incubated for 20 min at room temperature. For supershift assays, 0.2 μg of anti-NF-κB subunit antibodies were included in the reaction mixture. Samples were separated on native 6% polyacrylamide gels (Novex) in low ionic strength buffer (0.25 × Tris-borate-EDTA). Dried gels were exposed to a storage phosphor screen (Kodak) overnight and quantitated using a PhosphorImager (Molecular Dynamics). 10-cm dishes of HepG2 cells (approximately 80% confluent) or post-confluent fibroblasts were pre-incubated for 30 min in serum-free RPMI 1640 medium lacking phosphate and containing 20 mm HEPES, pH 7.4. This medium was then replaced with 3 ml/dish of the same medium supplemented with 0.4–1.0 mCi/ml [32P]orthophosphoric acid and incubation continued for 4 h. IL-1 was added to a final concentration of 20 ng/ml at the end of the labeling period in a minimal volume (less than 0.5%) of phosphate-free medium. Addition of vehicle alone had no effect on any of the reported parameters. After stimulation, cell monolayers were rinsed three times with ice-cold phosphate-buffered saline and then lysed in 1 ml of a buffer containing 50 mmTris-HCl, pH 8.0, 1% Nonidet P-40, 150 mm NaCl, 20 mm β-glycerophosphate, 10 mm NaF, 2 mm EGTA, 1 mm Na3VO4, 50 μm leupeptin, 50 μm pepstatin A, 1 mm PMSF (IP buffer). Lysates were passed 10 times through a 25 gauge hypodermic needle, incubated on ice for 10 min, then centrifuged at 13,000 × g for 15 min. Clarified lysates were diluted 2-fold into IP buffer supplemented with 0.2% SDS and 1% sodium deoxycholate. 1-ml aliquots were pre-cleared for 30 min with 20 μl of agarose-conjugated rabbit IgG (Sigma), and the supernatants were mixed overnight with 20 μl of a suspension of anti-p65-agarose (2.5 μg of antibody). Control incubations were carried out in the presence of a 40-fold molar excess of immunizing peptide. Immunoprecipitates were washed four times with IP buffer containing 0.1% SDS and 0.5% deoxycholate, twice with IP buffer containing 0.5 m NaCl, and once with 50 mmTris-HCl, pH 7.0. They were then resuspended in Laemmli sample buffer (24Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205531) Google Scholar), boiled for 5 min, and resolved by electrophoresis on 8–16% SDS-polyacrylamide gels (SDS-PAGE). The gels were dried and quantitated by phosphorimaging. In some cases, labeling with [32P]orthophosphate was omitted, and the washed NF-κB immunoprecipitates were further processed for measurement of associated casein kinase activity as described below. To determine casein kinase activity associated with cytoplasmic and nuclear NF-κB, cytoplasmic and nuclear extracts were prepared from IL-1-treated HepG2 cells as described for EMSA above except that buffers included phosphatase inhibitors (20 mm β-glycerophosphate, 10 mmNaF, 0.1 mm Na3VO4). 1-ml aliquots of both types of extract were adjusted to contain 1% Nonidet P-40, 0.1% SDS, and 500 mm NaCl in a final volume of 1.4 ml and were then immunoprecipitated with anti-p65-agarose as described above. Before assay, NF-κB immunoprecipitates were washed twice with 50 mm Tris-HCl, pH 7.5, 500 mm LiCl, 0.5 mm dithiothreitol, 20 mm β-glycerophosphate, 5 mm NaF, then twice with 50 mm Tris-HCl, pH 7.5, 20 mm β-glycerophosphate, 15 mmMgCl2, 5 mm NaF, 0.5 mmdithiothreitol. Purified casein kinase II (15 ng/reaction) was diluted in 15 μl of 50 mm Tris-HCl, pH 7.5, 1 mmdithiothreitol, 2 mm EDTA, 10% glycerol. Phosphorylation reactions were initiated by adding 30 μl of a kinase assay buffer containing 50 mm Tris-HCl, pH 7.5, 100 mm KCl, 15 mm MgCl2, 30 μm ATP, 2.5–4.5 μCi [γ-32P]ATP, 1 mm dithiothreitol, and substrate (dephosphorylated α- and β-caseins (Sigma) at 0.5 mg/ml or recombinant NF-κB p65 at approximately 5 μg/ml). When present, heparin was included at 20 μg/ml. Samples were incubated for 20 min at 30 °C with frequent mixing, and the reactions were terminated by addition of 10 μl of 4 × Laemmli sample buffer. Incorporation of labeled ATP into substrate proteins was visualized and quantitated by SDS-PAGE and phosphoimaging. For determination of total cellular casein kinase II activity, cells were treated with IL-1 as necessary, washed in PBS, and extracted in 50 mm HEPES, pH 7.9, 500 mm NaCl, 1% Triton X-100, 0.1% SDS, 20 mmβ-glycerophosphate, 10 mm NaF, 1 mm EGTA, 1 mm EDTA, 1 mm dithiothreitol, 1 mmPMSF, 50 μm pepstatin A, 50 μm leupeptin. After centrifugation (13,000 × g, 15 min), lysates were precleared with agarose-conjugated rabbit-IgG and then mixed for 4 h at 4 °C with a mixture of polyclonal antisera raised against human casein kinase II α- and β-subunits (1:200 dilution). Immunoprecipitates were collected on protein A-agarose (Sigma), washed three times with extraction buffer, and then processed for casein kinase assay as described above. Alternatively, confluent cells in 175-cm2 flasks (typically 4–10 flasks/treatment group) were treated with or without IL-1 for 15 min, washed with cold PBS, and scraped into lysis buffer (20 mm Tris-HCl, pH 8.5, 20 mm β-glycerophosphate, 50 mm NaF, 0.1 mm Na3VO4, 2 mmdithiothreitol, 0.5 mm EDTA, 0.5 mm EGTA, 0.1% Nonidet P-40, 1 mm PMSF, 50 μm pepstatin A, 50 μm leupeptin). The cell suspensions were lysed by passage through a 25 gauge hypodermic needle, incubated at 0 °C for 15 min, and clarified by centrifugation (25,000 × g, 15 min). Lysates were immediately loaded onto a Mono-Q HR 5/5 anion exchange column (Pharmacia) equilibrated in lysis buffer lacking Nonidet P-40 and containing only 10 mm NaF. The column was washed with 10 volumes of equilibration buffer and eluted with a gradient of 0–0.5 m NaCl in equilibration buffer at a flow rate of 1 ml min−1. Thirty 1-ml fractions were collected, and 10-μl aliquots of the fractions were assayed for casein kinase activity. Samples of radiolabeled α- and β-caseins or NF-κB p65 were eluted from polyacrylamide gel slices, TCA-precipitated, and oxidized (25Boyle W.J. Van Der Geer P. Hunter T. Methods Enzymol. 1991; 201: 110-149Crossref PubMed Scopus (1272) Google Scholar). Casein samples, in 100 μl of 50 mmNH4HCO3, were proteolytically digested by addition of 2 μg of sequencing grade chymotrypsin (Boehringer Mannheim). After 8 h, a second aliquot of chymotrypsin was added and digestion continued for 18 h. NF-κB p65 was incubated, as above, with 1 μg of chymotrypsin for 5 h, after which 1 μg of sequencing grade modified trypsin (Promega) was added, and incubation was continued for 18 h. The resulting peptides were washed several times by lyophilization from distilled water and applied to thin-layer cellulose plates for electrophoresis at pH 1.9 (1.0 kV for 45 min) followed by ascending chromatography in n-butyl alcohol:pyridine:acetic acid:water (7.5:5:1.5:6) for 6 h. Radiolabeled phosphoamino acid compositions were determined by partial acid digestion of gel-purified proteins followed by two-dimensional electrophoresis as described (26Hunter T. Sefton B. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 1311-1315Crossref PubMed Scopus (1540) Google Scholar). Cellular extracts were separated on 10% SDS-polyacrylamide gels (Novex). Following SDS-PAGE, proteins were electrophoretically transferred to nitrocellulose. After overnight blocking in Tris-buffered saline containing 5% non-fat dried milk and 0.1% Tween-20, blots were incubated with anti-NF-κB p65 (diluted to 2 μg/ml in Tris-buffered saline containing 1% non-fat dried milk) for 1 h. Immune complexes were detected by sequential incubation of the blots with horseradish peroxidase-conjugated goat anti-rabbit IgG (Jackson Laboratories) and visualized using ECL reagent (Amersham Corp.) coupled with autoradiography on X-Ray films (XAR-5, Kodak). The aim of this study was to determine if stimulation of HepG2 hepatoma cells or human gingival fibroblasts (HGF) with IL-1 leads to phosphorylation of other components of NF-κB besides the IκB inhibitor family. In both of these cell types, as shown by EMSA supershift analysis (Fig. 1), all of the IL-1-inducible nuclear NF-κB complexes contain p65, and a majority contain p50. After treatment with IL-1 for various times, lysates from cells metabolically labeled with [32P]orthophosphate were prepared and immunoprecipitated with anti-p65 antibody. To control for the presence of nonspecifically recognized proteins, we carried out duplicate immunoprecipitations, one of which contained an aliquot of the peptide against which the anti-p65 antibody was raised. As reported by others (16Li C.-C. Korner M. Ferris D.K. Chen E. Dai R.-M. Longo D.L. Biochem. J. 1994; 303: 499-506Crossref PubMed Scopus (40) Google Scholar), NF-κB/Rel family members and the IκBs are physically associated phosphoproteins and are readily co-precipitated as shown in Fig. 2. From lysates of untreated HepG2 cells and (less prominently) from HGF, a phosphoprotein of 40 kDa was specifically immunoprecipitated. We performed Western blot analysis with a specific antibody (not shown), and we found that this phosphoprotein corresponded to IκBα. After 5 min of IL-1 treatment, the 40-kDa species disappeared and was replaced by a slightly slower migrating species (shown as IκB(P) in Fig. 2) which also reacted with anti-IκBα. At subsequent times, IκB was undetectable as either a labeled phosphoprotein (Fig. 2) or by Western blotting (not shown). These data are entirely consistent with previous reports (27Beg A.A. Finco T.S. Nantermet P.V. Baldwin A.J. Mol. Cell. Biol. 1993; 13: 3301-3310Crossref PubMed Google Scholar, 28Cordle S.R. Donald R. Read M.A. Hawiger J. J. Biol. Chem. 1993; 268: 11803-11810Abstract Full Text PDF PubMed Google Scholar) in which activators of NF-κB were shown to increase the phosphorylation of IκB and thereby decrease its electrophoretic mobility. A protein that we tentatively identify as NF-κB p50 was transiently phosphorylated in both cell types following IL-1 treatment. Interestingly, Li et al. (16Li C.-C. Korner M. Ferris D.K. Chen E. Dai R.-M. Longo D.L. Biochem. J. 1994; 303: 499-506Crossref PubMed Scopus (40) Google Scholar) have reported that p50 phosphorylation was increased by treatment of Jurkat cells with phorbol 12-myristate 13-acetate and phytohemagglutinin and that phosphorylated p50 exhibited more stable DNA binding than the unphosphorylated form. Our most striking observation was an IL-1-mediated increase in the level of p65 phosphorylation. Although barely detectable in unstimulated HepG2 cells or fibroblasts, p65 was the most prominent phosphorylated species in immunoprecipitates made from cells treated with IL-1 for 5 min. Phosphorylation of P65 was maximal after 15 min and still evident for as long as 60 min after cytokine addition (not shown). IL-1-stimulated phosphorylation of an unidentified co-precipitated protein of about 130 kDa was consistently observed in HepG2 cells but was not investigated further. We isolated p65 from IL-1 stimulated HepG2 cells for phosphoamino acid analysis and found that phosphorylation occurred exclusively upon serine residues (Fig.3).Figure 2IL-1-dependent phosphorylation of NF-κB. HepG2 cells (top panel) and gingival fibroblasts (bottom panel) were metabolically labeled with [32P]orthophosphate and stimulated with 20 ng/ml IL-1 for the indicated times. NF-κB and associated proteins were immunoprecipitated in the absence (−) or presence (+) of excess immunizing peptide and visualized by phosphoimaging. Migration positions of 14C-labeled molecular weight standards (Amersham Corp.) are indicated on the right.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Basal and IL-1-mediated phosphorylation of NF-κB p65 occurs on serine residues. 32P-labeled p65 from untreated HepG2 cells (top panels) or cells treated for 15 min with IL-1 (bottom panels) was carefully excised from SDS-PAGE gels, extracted, and hydrolyzed. Samples were subjected to two-dimensional phosphoamino acid analysis and visualized using a PhosphorImager (left panels). Internal phosphoamino acid standards were detected using ninhydrin and are shown in the panels on the right (PS, phosphoserine; PT, phosphothreonine; PY, phosphotyrosine).View Large Image Figure ViewerDownload Hi-res image Download (PPT) IL-1 is known to activate a number of cytosolic serine/threonine kinases, including members of the mitogen-activated protein kinase family (20Bird T.A. Sleath P.R. deRoos P.C. Dower S.K. Virca G.D. J. Biol. Chem. 1991; 266: 22661-22670Abstract Full Text PDF PubMed Google Scholar), stress-activated protein kinase/p38/JNK family (21Litchfield D.W. Lozeman F.J. Cicirelli M.F. Harrylock M. Ericsson L.H. Piening C.J. Krebs E.G. J. Biol. Chem. 1991; 266: 20380-20389Abstract Full Text PDF PubMed Google Scholar,29Freshney N. Rawlinson L. Guesdon F. Jones E. Cowley S. Hsuan J. Saklatvala J. Cell. 1994; 78: 1039-1049Abstract Full Text PDF PubMed Scopus (773) Google Scholar, 30Kracht M. Truong O. Totty N.F. Shiroo M. Saklatvala J. J. Exp. Med. 1994; 180: 2017-2025Crossref PubMed Scopus (59) Google Scholar, 31Sluss H. Barrett T. Dérijard B. Davis R.J. Mol. Cell. Biol. 1994; 14: 8376-8384Crossref PubMed Scopus (444) Google Scholar), and a novel β-casein kinase (32Guesdon F. Freshney N. Waller R.J. Rawlinson L. Saklatvala J. J. Biol. Chem. 1993; 268: 4236-4243Abstract Full Text PDF PubMed Google Scholar, 33Guesdon F. Waller R.J. Saklatvala J. Biochem. J. 1994; 304: 761-768Crossref PubMed Scopus (26) Google Scholar). One of the preferred phosphorylation sites of the β-casein kinase purified from lungs of IL-1-injected rabbits is serine 57 of β-casein (34Guesdon F. Knight C.J. Saklatvala J. Cytokine. 1995; 7 (Abstr. 48): 603Google Scholar). 2G. D. Virca and T. A. Bird, unpublished observations. ) The primary sequence surrounding this residue bears some resemblance to a sequence in human p65 immediately COOH-terminal to the Rel-homology domain, surrounding a serine at position 340. Specifically, both sequences have prolines at the −6, +4, +6, +8, and +10 positions relative to the serine residue and hydrophobic residues at the −8 and +1 positions. p65 also contains two potential serine phosphorylation sites (35Kuenzel E.A. Mulligan J.A. Sommercorn J. Krebs E.G. J. Biol. Chem. 1987; 262: 9136-9140Abstract Full Text PDF PubMed Google Scholar) for casein kinase II (minimal recognition sequence: Ser [Thr]-X-X-Glu [Asp]) located at amino acid positions 276 and 539. We reasoned that the IL-1-stimulated kinase present in NF-κB immunoprecipitates might be one of these activities and that if it remained complexed with NF-κB during immunoprecipitation, it should be possible to detect it based on its ability to phosphorylate exogenously added casein. Fig.4 a shows that extensively washed p65 immunoprecipitates from untreated HepG2 cells contain a kinase activity capable of phosphorylating both α- and β-caseins and that the level of this activity is markedly stimulated if the cells are first treated with IL-1. The association of kinase activity with NF-κB in the immunoprecipitates is specific since it is prevented by inclusion of excess immunizing peptide in the immunoprecipitation mixtures (Fig. 4 a). Notably, kinase activity was completely abolished when the reactions were performed in the presence of 20 μg/ml heparin, a potent and characteristic inhibitor of casein kinase II (36Hathaway G.M. Lubben T.H. Traugh J.A. J. Biol. Chem. 1980; 255: 8038-8041Abstract Full Text PDF PubMed Google Scholar). None of the co-precipitated kinase activity could, therefore, be attributable to the cytokine-activated β-casein kinase, which is not inhibited by heparin at these concentrations and selectively phosphorylates β-casein (32Guesdon F. Freshney N. Waller R.J. Rawlinson L. Saklatvala J. J. Biol. Chem. 1993; 268: 4236-4243Abstract Full Text PDF PubMed Google Scholar). 3T. A. Bird and G. D. Virca, unpublished data α- and β-caseins phosphorylated by the co-precipitated kinase were subjected to chymotryptic digestion and two-dimensional phosphopeptide mapping. These maps were compared with maps of casein phosphorylated by authentic casein kinase II (Fig. 4 b). Although some of the digestion products (particularly in the case of α-casein) remained at the origin, the resolvable phosphopeptides were found to be essentially identical. Both kinases phosphorylated α-casein predominantly on serine and β-casein on threonine residues, with less phosphorylation on serine (data not shown). Taken together, these data confirm the identity of the NF-κB-associated kinase as casein kinase II. Because the anti-p65 antibody co-precipitates p50, and possibly other, associated proteins, it is not possible to determine from our data if p65 directly associates with the kinase. It is perhaps notable that the primary sequence of human p50 (37Kieran M. Blank V. Logeat F. Vandekerckhove J. Lottspeich F. Le Bail O. Urban M.B. Kourilsky P. Baeuerle P.A. Israël A. Cell. 1990; 62: 1007-1018Abstract Full Text PDF PubMed Scopus (601) Google Scholar) also contains a potential casein kinase II phosphorylation site (SDLE at position 338–341) that is conserved in both the rabbit and mouse p50 genes (38Ghosh S. Gifford A.M. Riviere L.R. Tempst P. Nolan G.P. Baltimore D. Cell. 1990; 62: 1019-1029Abstract Full Text PDF PubMed Scopus (587) Google Scholar). It was important to determine if the kinase associated with NF-κB in cell extracts was the same as that responsible for its phosphorylation in intact cells. Accordingly, we compared the phosphopeptide" @default.
• W2144639685 created "2016-06-24" @default.
• W2144639685 creator A5009580916 @default.
• W2144639685 creator A5023474363 @default.
• W2144639685 creator A5025935855 @default.
• W2144639685 creator A5047756694 @default.
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• W2144639685 date "1997-12-01" @default.
• W2144639685 modified "2023-10-14" @default.
• W2144639685 title "Activation of Nuclear Transcription Factor NF-κB by Interleukin-1 Is Accompanied by Casein Kinase II-mediated Phosphorylation of the p65 Subunit" @default.
• W2144639685 cites W1480839249 @default.
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